a. The role of HECT-ubiquitin ligases in HTLV budding. The process of particle morphogenesis and release is highly orchestrated involving viral components and cellular factors that have been recruited via small peptide motifs on viral proteins to perform functions necessary for the virus. We have previously shown that HTLV-1 like other retroviruses uses the ESCRT machinery of the cell for viral release and recruits ESCRT components via the so called late domainmotifs (LD). The PPPY LD motif in the matrix region of Gag recruits a ubiquitin ligase belonging to the HECT ubiquiting ligase (HUL) family. We have identified ITCH as the HUL most active in promoting HTLV-1 release We are currently further defining the role of different ITCH domains in budding and are analyzing the interactions between ITCH and components of the ESCRT system. b. Motifs in TM involved in Env trafficking. The transmembrane domain (TM) of HTLV-1 Env contains a YXXL motif and a PDZ-binding site. We have shown that both motifs are important in Env cellular trafficking and turnover thus affecting the infectivity of the virus particles. The YXXL motif interacts with adaptor proteins involved in the formation of endocytic vesicles. Perturbation of the motif results in stabilization of Env on the plasma membrane and increased fusogenicity. Mutating the PDZ-binding site causes increased endocytosis resulting in lower levels of Env on the cell surface. In collaboration with Dr. Walther Mothes (Yale University School of Medicine), we are copurifying Env complexes to identify the PDZ domain containing protein(s). c. Cell-to-cell transmission of HTLV-1 circumvents tetherin restriction. Tetherin is part of the cellular innate immunity and acts on many viruses. We have shown that like in the case of HIV, it strongly impairs release of viral particles from cells inhibiting cell-free infection by HTLV-1. Unlike HIV, which has evolved a function in the Vpu protein to counteract the tetherin effect, HTLV-1 seems to have not means to overcome this blockade to cell free transmission. However, cell-to-cell transmission of HTLV-1 is only minimally affected by tetherin. Indeed, viral particles and tetherin co-locate in the zone of cell-cell contact. We also showed that Tax expression is necessary for directing the trafficking of viral components to the site of cell-cell contact and enhances cell-to-cell transmission. d. Replication-dependent reporter vectors. We previously developed replication-dependent retroviral vectors to study cell-to-cell transmission. These vectors have reporter gene that are silent in the producer cell. The reporters are activated (by splicing and reverse transcription) producing a signal in the target cell. We began by generating HTLV-1 and HIV-1 vectors. We have since produced similar constructs for HTLV-2,-3 and 4, murine leukemia virus (MLV), squirrel monkey retrovirus type 1 (SMRV-1), and simian retrovirus type 1 (SRV-1). We are still developing vectors for simian immunodeficiency virus (SIV) and bovine leukemia virus (BLV). We have also produced a second generation of reporters for use in all the retroviral vector. These reporters have about a 50-fold increase in the signal which makes them suitable for use in high-throughput assays. We are currently developing susceptible cell lines with stably integrated HIV-1 reporter constructs to measure the replicative potential of patient samples in high-throughput assays. e. To better understand HTLV-1 pathogenesis and persistence, we are constructing molecular clones and vectors for the newly discovered human deltaretroviruses HTLV-3 and HTLV-4 to take advantage of the biological and genetic diversity among these viruses for comparative analyses. HTLV-3 and HTLV-4 are recent isolates and were isolated several times independently from geographically separated individuals. However, in all cases the people had direct contact with primates, which could have been infected with similar viruses. There was no evidence of human to human transmission. We have obtained the viral genome sequences for both strains on several PCR fragments and have reconstructed infectious proviruses. The HTLV-3 proviral clone is fully active and can initiate spreading infections in B5 cells. The HTLV-4 proviral clone is active in single-round infection experiments but cannot yet sustain a spreading infection. This project was done in collaboration with William Switzer at the Centers for Disease Control and Prevention.